Small chemicals like drugs tend to bind to proteins via noncovalent bonds, e.g. hydrogen bonds, salt bridges or electrostatic interactions. Some chemicals interact with other molecules than the actual target ligand, representing so-called ‘off-target' activities of drugs. Such interactions are a main cause of adverse side effects to drugs and are normally classified as predictable type A reactions. Detailed analysis of drug-induced immune reactions revealed that off-target activities also affect immune receptors, such as highly polymorphic human leukocyte antigens (HLA) or T cell receptors (TCR). Such drug interactions with immune receptors may lead to T cell stimulation, resulting in clinical symptoms of delayed-type hypersensitivity. They are assigned the ‘pharmacological interaction with immune receptors' (p-i) concept. Analysis of p-i has revealed that drugs bind preferentially or exclusively to distinct HLA molecules (p-i HLA) or to distinct TCR (p-i TCR). P-i reactions differ from ‘conventional' off-target drug reactions as the outcome is not due to the effect on the drug-modified cells themselves, but is the consequence of reactive T cells. Hence, the complex and diverse clinical manifestations of delayed-type hypersensitivity are caused by the functional heterogeneity of T cells. In the abacavir model of p-i HLA, the drug binding to HLA may result in alteration of the presenting peptides. More importantly, the drug binding to HLA generates a drug-modified HLA, which stimulates T cells directly, like an allo-HLA. In the sulfamethoxazole model of p-i TCR, responsive T cells likely require costimulation for full T cell activation. These findings may explain the similarity of delayed-type hypersensitivity reactions to graft-versus-host disease, and how systemic viral infections increase the risk of delayed-type hypersensitivity reactions.

Abacavir hypersensitivity is a severe hypersensitivity reaction which occurs exclusively in carriers of the HLA-B*57∶01 allele. In vitro culture of PBMC with abacavir results in the outgrowth of abacavir-reacting CD8+ T cells, which release IFNγ and are cytotoxic. How this immune response is induced and what is recognized by these T cells is still a matter of debate. We analyzed the conditions required to develop an abacavir-dependent T cell response in vitro. The abacavir reactivity was independent of co-stimulatory signals, as neither DC maturation nor release of inflammatory cytokines were observed upon abacavir exposure. Abacavir induced T cells arose in the absence of professional APC and stemmed from naïve and memory compartments. These features are reminiscent of allo-reactivity. Screening for allo-reactivity revealed that about 5% of generated T cell clones (n = 136) from three donors were allo-reactive exclusively to the related HLA-B*58∶01. The addition of peptides which can bind to the HLA-B*57∶01-abacavir complex and to HLA-B*58∶01 during the induction phase increased the proportion of HLA-B*58∶01 allo-reactive T cell clones from 5% to 42%. In conclusion, abacavir can alter the HLA-B*57∶01-peptide complex in a way that mimics an allo-allele (‘altered self-allele’) and create the potential for robust T cell responses.

Drug-induced liver injury (DILI) is a main cause of drug withdrawal. A particularly interesting example is flucloxacillin (FLUX)-DILI, which is associated with the HLA-B*57:01 allele. At present, the mechanism of FLUX-DILI is not understood, but the HLA association suggests a role for activated T cells in the pathomechanism of liver damage. To understand the interaction among FLUX, HLA molecules, and T cells, we generated FLUX-reacting T cells from FLUX-naive HLA-B*57:01+ and HLA-B*57:01− healthy donors and investigated the mechanism of T cell stimulation. We found that FLUX stimulates CD8+ T cells in two distinct manners. On one hand, FLUX was stably presented on various HLA molecules, resistant to extensive washing and dependent on proteasomal processing, suggesting a hapten mechanism. On the other hand, in HLA-B*57:01+ individuals, we observed a pharmacological interaction with immune receptors (p-i)–based T cell reactivity. FLUX was presented in a labile manner that was further characterized by independence of proteasomal processing and immediate T cell clone activation upon stimulation with FLUX in solution. This p-i–based T cell stimulation was restricted to the HLA-B*57:01 allele. We conclude that the presence of HLA-B*57:01 drives CD8+ T cell responses to the penicillin-derivative FLUX toward nonhapten mechanism.

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Rationaleβ-lactam antibiotics cause drug hypersensitivity reactions (DHR) with various clinical pictures from minor affections like maculopapular exanthema (MPE) and urticaria to severe cutaneous adverse reactions and anaphylaxis. Currently, two different reactivity patterns have been shown to initiate an immune reaction by activating T cells—the hapten concept and the pharmacological interaction with immune receptor (p–i) concept.ObjectivesIn this study, the relationship between the reactivity pattern of drug-reacting T cells of drug allergic patients and their clinical picture has been investigated.FindingsDrug-reacting T-cell clones (TCCs) were isolated from patients hypersensitive to β-lactams. Analysis of their reactivity pattern revealed an exclusive use of the hapten mechanism for patients with immediate reactions and for patients of MPE. In patients suffering from drug reactions with eosinophils and systemic symptoms, a severe DHR, analysis of isolated drug-reacting TCC identified the p–i concept as the unique mechanism for T-cell activation.ConclusionsThe results show a shift from hapten pattern in mild allergic reactions to p–i pattern in severe life-threatening allergic reactions. They strongly argue against the current preclinical risk evaluation of new drugs based on the ability to form haptens.

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Rational: Drug induced liver injury (DILI) is a main cause of drug withdrawal. A particularly interesting example is flucloxacillin (FLUX)-DILI, which is associated with the HLA-B*5701 allele. To better understand this side effect, we established an in vitro model for FLUX induced T cell activation. Methods: Peripheral blood mononuclear cells from FLUX-naïve, HLA-B*5701+ healthy donors were induced by iterative in vitro restimulations with FLUX. FLUX-specific T cells (FLUX-Tc) were enriched by positive selection of CD107a expressing cells and characterized by flow cytometry and 51Cr-release assay. Results: FLUX-Tc are mainly CD8+. They react by IFNγ secretion and CD107a upregulation upon drug stimulation and are cytotoxic against autologous antigen presenting cells (APC). Cytotoxicity and reactivity was observed only if free FLUX was added to the system. FLUX-pulsed APC were not able to elicit a FLUX specific T cell response if FLUX was not additionally added. The direct addition of FLUX to FLUX-Tc resulted in their activation even in the absence of APC. This self-presentation and reactivation was still observed in the presence of the proteasome inhibitors lactacystin and bortezomib. Conclusion: The APC independent T cell reactivity and the non-involvement of the proteasome is best explained by a direct interaction of FLUX with the restricting HLA-B*5701 molecule. How and why this activation leads to a preferential damage of liver cells needs further investigation.